Polypeptides with lipase activity and polynucleotides encoding same for cleaning

ABSTRACT

Isolated polypeptides with lipase activity; polynucleotides encoding the polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the polypeptides for cleaning are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/104,383filed Jun. 14, 2016, now allowed, which is a 35 U.S.C. 371 nationalapplication of PCT/CN2015/070870 filed Jan. 16, 2015 which claimspriority or the benefit under 35 U.S.C. 119 of Chinese PCT applicationnos. PCT/CN2014/071149 and PCT/CN2014/071146 filed Jan. 22, 2014 andJan. 22, 2014, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides with lipase activity,polynucleotides encoding the polypeptides, methods of producing thepolypeptides, and methods of using the polypeptides.

Description of the Related Art

Lipases are important biocatalysts which have shown to be useful forvarious applications and a large number of different lipases have beenidentified and many commercialized. However, new lipases suitable foruse in various compositions adapted to conditions currently used aredesirable.

Lipases have been employed in compositions for the removal of lipidstains by hydrolyzing triglycerides to generate fatty acids. Currentcleaning and/or fabric care compositions comprise many activeingredients which are interfering with the ability of lipases to removelipid stains. Thus, the need exists for lipases that can function in theharsh environment of compositions used for cleaning. It is the object ofthe present application to provide lipases that has advantageouscharacteristics, such as improved performance when applied in cleaningcompositions.

A known lipase from Mucor circinelloides f. circinelloides (UNIPROT:S2JTM3) is 72% identical to the mature polypeptides of SEQ ID NO: 2 andSEQ ID NO: 6 of the present application.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides with lipaseactivity, selected from the group consisting of: (a) a polypeptidehaving at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) SEQ ID NO: 1 or SEQ ID NO: 5, or thefull-length complement of (i); (c) a polypeptide encoded by apolynucleotide having at least 75%,at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5; (d) a polypeptidewhich is a variant of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g. several) positions; and (e) apolypeptide which is a fragment of any of the polypeptides of (a), (b),(c) or (d).

The present invention also relates to isolated polynucleotides encodingthe polypeptides; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the polypeptideand use of the polypeptide.

The present invention also relates to compositions comprising thepolypeptide and methods of using the polypeptide for cleaning.

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino (N-) and/or carboxyl(C-) terminus of the mature polypeptide; wherein the fragment has lipaseactivity. In one aspect, the fragment contains at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 80%, at least85%, at least 90%, or at least 95% of the number of amino acids 1 to 356of SEQ ID NO: 2 or amino acids 1 to 351 of SEQ ID NO: 6.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, polypeptide,variant, nucleic acid, protein, peptide or cofactor, that is at leastpartially removed from one or more (e.g., several)or all of thenaturally occurring constituents with which it is associated in nature;(3) any substance modified by the hand of man relative to that substancefound in nature; or (4) any substance modified by increasing the amountof the substance relative to other components with which it is naturallyassociated (e.g., multiple copies of a gene encoding the substance; useof a stronger promoter than the promoter naturally associated with thegene encoding the substance). An isolated substance may be present in afermentation broth sample.

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC 3.1.1 as defined by Enzyme Nomenclature. It may havelipase activity (triacylglycerol lipase, EC 3.1.1.3), cutinase activity(EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13) and/or wax-esterhydrolase activity (EC 3.1.1.50). For purposes of the present invention,lipase activity is determined according to the procedure described inthe Examples. In one aspect, the variants of the present invention haveat least 20%, e.g., at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, or 100% of the lipase activity of the polypeptide of SEQ ID NO: 2or SEQ ID NO: 6.

Low temperature: “Low temperature” is a temperature of 5-35° C., such as5-30° C., 5-25° C., 5-20° C., 5-15° C., or 5-10° C. In anotherembodiment, “Low temperature” is a temperature of 10-35° C., such as10-30° C., 10-25° C., 10-20° C., or 10-15° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 356 of SEQ ID NO: 2 or amino acids 1 to351 of SEQ ID NO: 6 based on the program predicting the signal peptide,e.g., SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6)] thatpredicts amino acids −27 to 1 of SEQ ID NO: 2 or amino acids −27 to 1 ofSEQ ID NO: 6 are a signal peptide. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is comprised in the nucleotides 82 to 1149 of SEQ ID NO: 1 orthe nucleotides 82 to 1134 of SEQ ID NO: 5 based on the programpredicting the signal peptide, e.g., SignalP (Nielsen et al., 1997,supra)] that predicts nucleotides 1 to 81 of SEQ ID NO: 1 or nucleotides1 to 81 of SEQ ID NO: 5 encodes a signal peptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which a substitution is made to produce the lipase variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: Very low stringency conditions: The term “verylow stringency conditions” means for probes of at least 100 nucleotidesin length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 25%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 45° C. Low stringency conditions: Theterm “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C. Medium stringencyconditions: The term “medium stringency conditions” means for probes ofat least 100 nucleotides in length, prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 55° C.Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/mL sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C. High stringency conditions: Theterm “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C. Very high stringencyconditions: The term “very high stringency conditions” means for probesof at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lipase activity. In one aspect, a subsequence containsat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 80%, at least 85, at least 90%, and at least 95% of the number ofnucleotides of 82 to 1149 of SEQ ID NO: 1 or nucleotides 82 to 1134 ofSEQ ID NO: 5

Variant: The term “variant” means a polypeptide having lipase activitycomprising a substitution at one or more (e.g., several) positions i.e.a variant of the present invention is also a polypeptide of the presentinvention. A substitution means replacement of the amino acid occupyinga position with a different amino acid. The variants of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the lipase activity of SEQ ID NO: 2 or SEQ ID NO: 6.

Wash performance: In the present context the term “wash performance” isused as an enzyme's ability to remove lipid or lipid-containing stainspresent on the object to be cleaned. The wash performance may bequantified by calculating the so-called G/(B+R) value defined in thedescription of AMSA in the Methods section below. The term “washperformance” includes cleaning in general e.g. hard surface cleaning asin dish wash, but also wash performance on textiles such as laundry, andalso industrial and institutional cleaning.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature. A “wild-type” lipase may berecominantly expressed in a host cell.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides with Lipase Activity

In an embodiment, the present invention relates to isolated polypeptideswith lipase activity, selected from the group consisting of: (a) apolypeptide having at least 75%,at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQ IDNO: 6; (b) a polypeptide encoded by a polynucleotide that hybridizesunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) SEQ ID NO: 1 or SEQ ID NO: 5, or thefull-length complement of (i); (c) a polypeptide encoded by apolynucleotide having at least 75%,at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5; (d) a polypeptidewhich is a variant of SEQ ID NO: 2 or SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (e.g. several)positions; and (e) a polypeptide which is a fragment of any of thepolypeptides of (a), (b), (c) or (d).

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or an allelicvariant thereof; or is a fragment thereof having lipase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO:6. In another aspect, thepolypeptide comprises or consists of amino acids 1 to 356 of SEQ ID NO:2 or amino acids 1 to 351 of SEQ ID NO: 6.

In another embodiment, the present invention relates to an isolatedpolypeptide having lipase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or SEQ ID NO: 5, or the full-length complement of (i) (Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.).

In another aspect, the polypeptide is a fragment of the polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 6. The fragment may contain at least 250amino acid residues, e.g., at least 255, at least 260, at least 265, atleast 270, at least 275, at least 280, at least 285, at least 290, atleast 295, at least 300, at least 305, at least 310, at least 315, atleast 320, at least 325, at least 330, at least 335, at least 340, atleast 345, or at least 350 amino acid residues. In one aspect, thefragment contains at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% of the number of amino acids of the polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 6.

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, or thepolynucleotide of SEQ ID NO: 5 or a subsequence thereof, as well as thepolypeptide of SEQ ID NO: 6 or a fragment thereof may be used to designnucleic acid probes to identify and clone DNA encoding polypeptideshaving lipase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic DNA or cDNA of a cell ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having lipase activity. Genomic or other DNA fromsuch other strains may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1, SEQ ID NO: 5 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1 or SEQ ID NO: 5; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 5; (iii) the full-lengthcomplement thereof; or (iv) a subsequence thereof; under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is SEQ ID NO: 1 or SEQ ID NO: 5.In another aspect, the nucleic acid probe consists of at least 15 and upto 1000 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 5. In another aspect,the nucleic acid probe is a polynucleotide that encodes the polypeptideof SEQ ID NO: 2 or SEQ ID NO: 6; or a fragment thereof.

In another embodiment, the present invention relates to an isolatedpolypeptide having lipase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 5 of at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to variants of thepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 6, of the mature polypeptideof SEQ ID NO: 2 or SEQ ID NO: 6, or of a fragment thereof comprising asubstitution at one or more (e.g., several) positions. In an embodiment,the number of amino acid substitutions introduced into the polypeptideof SEQ ID NO: 2 or SEQ ID NO: 6 is 1-50, 1-40, 1-30, 1-20, 1-10 or 1-5,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

It is contemplated that the polypeptide of the invention as describedabove may also provide basis for one or more (e.g. several)substitutions for generation of lipase variants. Accordingly thepolypeptide will also be a parent lipase.

Sources of Polypeptides with Lipase Activity

The polypeptide with lipase activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptide issecreted extracellularly.

The polypeptide may be a bacterial lipase. For example, the polypeptidemay be a Gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyceslipase, ora Gram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis lipase.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus lipase.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans lipase.

The polypeptide may be a fungal lipase. For example, the polypeptide maybe a yeast lipase such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia lipase; or a filamentousfungal lipase such as an Acremonium, Agaricus, Alternaria, Aspergillus,Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Nectria, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria lipase.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis lipase.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Mucor circinelloides, Myceliophthorathermophila, Neurospora crassa, Penicillium funiculosum, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia setosa, Thielavia spededonium, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride lipase.

In another aspect, the polypeptide is an Actinomucor e/egans lipase,e.g., the lipase of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO:2, or a fragment thereof. In another aspect, the polypeptide is anActinomucor elegans lipase, e.g., the lipase of SEQ ID NO: 6, the maturepolypeptide of SEQ ID NO: 6, or a fragment thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding apolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Preparation of Polypeptides

The present invention relates to methods for obtaining a polypeptidehaving lipase activity, comprising: (a) introducing into a parent lipase(e.g. a wild type) a substitution at one or more (e.g., several)positions corresponding to positions of the polypeptide of SEQ ID NO: 2;and (b) recovering the variant. The present invention also relates tomethods for obtaining a polypeptide having lipase activity, comprising:(a) introducing into a parent lipase (e.g. a wild type) a substitutionat one or more (e.g., several) positions corresponding to positions ofthe polypeptide of SEQ ID NO: 6; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the polypeptide. The promoter may be any polynucleotidethat shows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the polypeptide. Any terminator that is functional in the hostcell may be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus nigerglucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of agene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the polypeptide. Any leader that is functional in the host cellmay be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polypeptide-encoding sequenceand, when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of thepolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a polypeptide of the present inventionoperably linked to one or more control sequences that direct theproduction of a polypeptide of the present invention. A construct orvector comprising a polynucleotide is introduced into a host cell sothat the construct or vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Mucorcircinelliodes, Myceliophthora thermophila, Neurospora crassa,Penicillium purpurogenum, Phanerochaete chrysosporium, Phiebia radiata,Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing apolypeptide, comprising: (a) cultivating a host cell of the presentinvention under conditions suitable for expression of the polypeptide;and (b) recovering the polypeptide. In a preferred aspect, the cell isan Aspergillus cell. In a more preferred aspect, the cell is anAspergillus oryzae cell. In a most preferred aspect the cell isAspergillus oryzae MT3568.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide. Examplesof lipase activity assays are known in the art including plate assay andpNP assay as described in the in the examples.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Compositions

Compositions comprising the polypeptide of the present inventions arealso contemplated.

In certain aspects the present invention relates to isolatedpolypeptides with lipase activity, selected from the group consistingof: (a) a polypeptide having at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQID NO: 6; (b) a polypeptide encoded by a polynucleotide that hybridizesunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) SEQ ID NO: 1, SEQ ID NO: 5 or thefull-length complement of (i); (c) a polypeptide encoded by apolynucleotide having at least 75%,at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5; (d) a polypeptidewhich is a variant of SEQ ID NO: 2 or SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (e.g. several)positions; and (e) a polypeptide which is a fragment of any of thepolypeptides of (a), (b), (c) or (d).

In certain aspects the invention relates to compositions comprising apolypeptide, comprising a substitution at one or more (e.g., several)positions of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 2 or afragment thereof. In some aspects the invention relates to compositionscomprising a polypeptide, which is unchanged i.e. not comprising asubstitution at one or more (e.g., several) positions of SEQ ID NO: 2the mature polypeptide of SEQ ID NO: 2 or a fragment thereof. In certainaspects the invention relates to compositions comprising a polypeptide,comprising a substitution at one or more (e.g., several) positions ofSEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 6 or a fragmentthereof. In some aspects the invention relates to compositionscomprising a polypeptide, which is unchanged i.e. not comprising asubstitution at one or more (e.g., several) positions of SEQ ID NO: 6the mature polypeptide of SEQ ID NO: 6 or a fragment thereof.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain embodiments of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Unless otherwise indicated the amounts in percentage is by weight of thecomposition (wt %). Suitable component materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, enzymes, and enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, hueing dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents and/or pigments. In addition to the disclosure below,suitable examples of such other components and levels of use are foundin U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348 herebyincorporated by reference.

Thus, in certain embodiments the invention do not contain one or more ofthe following adjuncts materials: surfactants, soaps, builders,chelating agents, dye transfer inhibiting agents, dispersants,additional enzymes, enzyme stabilizers, catalytic materials, bleachactivators, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, perfume delivery systems, structure elasticizing agents,fabric softeners, carriers, hydrotropes, processing aids, solventsand/or pigments. However, when one or more components are present, suchone or more components may be present as detailed below:

Surfactants—The compositions according to the present invention maycomprise a surfactant or surfactant system wherein the surfactant can beselected from nonionic surfactants, anionic surfactants, cationicsurfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof. When present,surfactant is typically present at a level of from 0.1 to 60 wt %, from0.2 to 40 wt %, from 0.5 to 30 wt %, from 1 to 50 wt %, from 1 to 40 wt%, from 1 to 30 wt %, from 1 to 20 wt %, from 3 to 10 wt %, from 3 to5wt %, from 5 to 40 wt %, from 5 to 30 wt %, from 5 to 15wt %, from 3 to20 wt %, from 3 to 10 wt %, from 8 to 12wt %, from 10 to 12wt % or from20 to 25wt %. Suitable anionic detersive surfactants include sulphateand sulphonate detersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in another aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3. The alkyl sulphate, alkylalkoxylated sulphate and alkyl benzene sulphonates may be linear orbranched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Non-limiting examples of anionic surfactants include sulfates andsulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomersof LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof.

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄-C₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includeLutensol®.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), as well as products available underthe trade names SPAN and TWEEN, and combinations thereof.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺ X⁻, wherein, Ris a linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Non-limiting examples of cationic surfactants includealkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, ester quats, andcombinations thereof. Suitable amphoteric/zwitterionic surfactantsinclude amine oxides and betaines such as alkyldimethylbetaines,sulfobetaines, or combinations thereof. Amine-neutralized anionicsurfactants—Anionic surfactants of the present invention and adjunctanionic cosurfactants, may exist in an acid form, and said acid form maybe neutralized to form a surfactant salt which is desirable for use inthe present detergent compositions. Typical agents for neutralizationinclude the metal counterion base such as hydroxides, eg, NaOH or KOH.Further preferred agents for neutralizing anionic surfactants of thepresent invention and adjunct anionic surfactants or cosurfactants intheir acid forms include ammonia, amines, or alkanolamines.Alkanolamines are preferred. Suitable non-limiting examples includingmonoethanolamine, diethanolamine, triethanolamine, and other linear orbranched alkanolamines known in the art; e.g., highly preferredalkanolamines include 2-amino-1-propanol, 1-aminopropanol,monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization may bedone to a full or partial extent, e.g. part of the anionic surfactantmix may be neutralized with sodium or potassium and part of the anionicsurfactant mix may be neutralized with amines or alkanolamines.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide.

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

In certain embodiments of the invention the composition comprisessurfactants or surfactant systems selected from sodium dodecyl benzenesulfonate, sodium hydrogenated cocoate, sodium laureth sulfate, C12-14pareth-7, C12-15 pareth-7, sodium C12-15 pareth sulfate, and C14-15pareth-4.

Soap—The compositions herein may contain soap. Without being limited bytheory, it may be desirable to include soap as it acts in part as asurfactant and in part as a builder and may be useful for suppression offoam and may furthermore interact favorably with the various cationiccompounds of the composition to enhance softness on textile fabricstreaded with the inventive compositions. Any soap known in the art foruse in laundry detergents may be utilized. In one embodiment, thecompositions contain from 0 wt % to 20 wt %, from 0.5wt % to 20 wt %,from 4wt % to 10 wt %, or from 4wt % to 7wt % of soap.

Examples of soap useful herein include oleic acid soaps, palmitic acidsoaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soapsare in the form of mixtures of fatty acid soaps having different chainlengths and degrees of substitution. One such mixture is topped palmkernel fatty acid.

In one embodiment, the soap is selected from free fatty acid. Suitablefatty acids are saturated and/or unsaturated and can be obtained fromnatural sources such a plant or animal esters (e.g., palm kernel oil,palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil,tallow and fish oils, grease, and mixtures thereof), or syntheticallyprepared (e.g., via the oxidation of petroleum or by hydrogenation ofcarbon monoxide via the Fisher Tropsch process).

Examples of suitable saturated fatty acids for use in the compositionsof this invention include captic, lauric, myristic, palmitic, stearic,arachidic and behenic acid. Suitable unsaturated fatty acid speciesinclude: palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.Examples of preferred fatty acids are saturated Cn fatty acid, saturatedCi₂-Ci₄ fatty acids, and saturated or unsaturated Cn to Ci₈ fatty acids,and mixtures thereof.

When present, the weight ratio of fabric softening cationic cosurfactantto fatty acid is preferably from about 1:3 to about 3: 1, morepreferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.

Levels of soap and of nonsoap anionic surfactants herein are percentagesby weight of the detergent composition, specified on an acid form basis.However, as is commonly understood in the art, anionic surfactants andsoaps are in practice neutralized using sodium, potassium oralkanolammonium bases, such as sodium hydroxide or monoethanolamine.

Hydrotropes—The compositions of the present invention may comprise oneor more hydrotropes. A hydrotrope is a compound that solubiliseshydrophobic compounds in aqueous solutions (or oppositely, polarsubstances in a non-polar environment). Typically, hydrotropes have bothhydrophilic and a hydrophobic character (so-called amphiphilicproperties as known from surfactants); however the molecular structureof hydrotropes generally do not favor spontaneous self-aggregation, seee.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid &Interface Science 12: 121-128. Hydrotropes do not display a criticalconcentration above which self-aggregation occurs as found forsurfactants and lipids forming miceller, lamellar or other well definedmeso-phases. Instead, many hydrotropes show a continuous-typeaggregation process where the sizes of aggregates grow as concentrationincreases. However, many hydrotropes alter the phase behavior,stability, and colloidal properties of systems containing substances ofpolar and non-polar character, including mixtures of water, oil,surfactants, and polymers. Hydrotropes are classically used acrossindustries from pharma, personal care, food, to technical applications.Use of hydrotropes in detergent compositions allow for example moreconcentrated formulations of surfactants (as in the process ofcompacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt %, such as from 0 to 5wt %,0.5 to 5wt %, or from 3% to 5wt %, of a hydrotrope. Any hydrotrope knownin the art for use in detergents may be utilized. Non-limiting examplesof hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate(SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders—The compositions of the present invention may comprise one ormore builders, co-builders, builder systems or a mixture thereof. When abuilder is used, the cleaning composition will typically comprise from 0to 65wt %, at least 1wt %, from 2 to 60 wt % or from 5 to 10 wt %builder. In a dish wash cleaning composition, the level of builder istypically 40 to 65wt % or 50 to 65wt %. The composition may besubstantially free of builder; substantially free means “no deliberatelyadded” zeolite and/or phosphate. Typical zeolite builders includezeolite A, zeolite P and zeolite MAP. A typical phosphate builder issodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid)(ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO09/102854, U.S. Pat. No. 5,977,053.

In one aspect, the invention relates to compositions comprising isolatedpolypeptides with lipase activity, selected from the group consistingof: (a) a polypeptide having at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQID NO: 6; (b) a polypeptide encoded by a polynucleotide that hybridizesunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) SEQ ID NO: 1, SEQ ID NO: 5 or thefull-length complement of (i); (c) a polypeptide encoded by apolynucleotide having at least 75%,at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5; (d) a polypeptidewhich is a variant of SEQ ID NO: 2 or SEQ ID NO: 6 comprising asubstitution, deletion, and/or insertion at one or more (e.g. several)positions; and (e) a polypeptide which is a fragment of any of thepolypeptides of (a), (b), (c) or (d), the composition comprising up to10wt % or 15wt % aluminosilicate (anhydrous basis) and/or phosphatebuilder, the composition having a reserve alkalinity of greater than 4or 7.5. As used herein the term “reserve alkalinity” is a measure of thebuffering capacity of the composition (g/NaOH/100 g composition)determined by titrating a 1% (w/v) solution of composition withhydrochloric acid to pH 7.5 i.e. in order to calculate reservealkalinity. Reserve alkalinity may be calculated as disclosed on page 9in WO2006/090335.

Chelating Agents and Crystal Growth Inhibitors—The compositions hereinmay contain a chelating agent and/or a crystal growth inhibitor.Suitable molecules include copper, iron and/or manganese chelatingagents and mixtures thereof. Suitable molecules include DTPA (Diethylenetriamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),DTPMP (Diethylene triamine penta(methylene phosphonic acid)),1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid(EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005to 15 wt % or from 3.0 to 10 wt % chelating agent or crystal growthinhibitor.

Bleach Component—The bleach component suitable for incorporation in themethods and compositions of the invention comprise one or a mixture ofmore than one bleach component. Suitable bleach components includebleaching catalysts, photobleaches, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, pre-formed peracids and mixturesthereof. In general, when a bleach component is used, the compositionsof the present invention may comprise from 0 to 30 wt %, from 0.00001 to90 wt %, 0.0001 to 50 wt %, from 0.001 to 25wt % or from 1 to 20 wt %.Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular c-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt % or from 0.1 to 20 wt %.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt % or 1 to 30 wt % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

(3) The term bleach activator is meant herein as a compound which reactswith hydrogen peroxide to form a peracid via perhydrolysis. The peracidthus formed constitutes the activated bleach. Suitable bleach activatorsto be used herein include those belonging to the class of esters,amides, imides or anhydrides. Suitable bleach activators are thosehaving R—(C═O)-L wherein R is an alkyl group, optionally branched,having, when the bleach activator is hydrophobic, from 6 to 14 carbonatoms, or from 8 to 12 carbon atoms and, when the bleach activator ishydrophilic, less than 6 carbon atoms or less than 4 carbon atoms; and Lis leaving group. Examples of suitable leaving groups are benzoic acidand derivatives thereof—especially benzene sulphonate. Suitable bleachactivators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A family of bleach activators is disclosed in EP624154and particularly preferred in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmentally friendly. Furthermore acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally ATC ismultifunctional, as the citrate released in the perhydrolysis reactionmay function as a builder. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt %, 0.5 to 40 wt % or 0.6 to 10wt % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt %, or 0.1 to 20 wt %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g. —N⁺ (CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g. —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆-C₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both Wand R²are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of, theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred embodiment the DAP may be asymmetric, such thatpreferably the hydrolysis of R1 acyl group is rapid to generate peracid,but the hydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n-C(O)—OO—C(O)—R³, in which R³ represents aC₁-C₉alkyl, or C₃-C_(7,) group and n represents an integer from 2 to 12,or 4 to 10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredembodiment, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (e.g. compound 4, p.433);N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,569 (e.g. Column 11, Example 1); andN-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (e.g. Column 10, Ex. 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (e.g. Column 31, Ex. II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (e.g. Column 32, Ex. V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described inWO05/047264 (e.g. p.18, Ex. 8), and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of OrganicChemistry(1990),55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinic amide, which can be made according to the proceduresdescribed in the Journal of the Chemical Society, ChemicalCommunications (1994), (22), 2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in US5753599 (Column9, Ex. 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In another aspect, thedetergent composition comprises a bleach component having a logP_(o/w)no greater than 0, no greater than −0.5, no greater than −1.0, nogreater than −1.5, no greater than −2.0, no greater than −2.5, nogreater than −3.0, or no greater than −3.5. The method for determininglogP_(o/w) is described in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,CO₂ ⁻, OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety—CR¹¹R¹²—Y-G_(b)-Y_(c)—[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubsituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt %, from 0.5 to 40wt % or from 0.6 to 10 wt % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. Nos. 5,597,936; 5,595,967. Such cobalt catalysts arereadily prepared by known procedures, such as taught e.g. in U.S. Pat.Nos. 5,597,936 and 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Exemplary bleaching systems are also described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259 and WO2007/087242.

Fabric Hueinq Agents—The composition may comprise a fabric hueing agent.Suitable fabric hueing agents include dyes, dye-clay conjugates, andpigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Color Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Color Index (Society of Dyersand Colorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In another aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In another aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken), dye-polymer conjugates formedfrom at least one reactive dye and a polymer selected from the groupconsisting of polymers comprising a moiety selected from the groupconsisting of a hydroxyl moiety, a primary amine moiety, a secondaryamine moiety, a thiol moiety and mixtures thereof. In still anotheraspect, suitable polymeric dyes include polymeric dyes selected from thegroup consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC)conjugated with a reactive blue, reactive violet or reactive red dyesuch as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colorants, alkoxylatedthiophene polymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO08/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]  a)

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

R₁=alkyl, aryl, or aryl alkyl and R₂=[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]  b)

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

R₁=[CH₂CH₂(OR₃)CH₂OR₄] and R₂=[CH₂CH₂(OR₃)CH₂OR₄]  c)

wherein R₃ is selected from the group consisting of H, (CH₂CH₂O)_(z)H,and mixtures thereof; and

-   wherein z=0 to 10;-   wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl,    aryl groups, and mixtures thereof; and

d) wherein R1 and R2 can independently be selected from the aminoaddition product of styrene oxide, glycidyl methyl ether, isobutylglycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by theaddition of from 1 to 10 alkylene oxide units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 1 R1 R2 R′ R″ x y R′ R″ x y a H H 3 1 H H 0 1 b H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1Further whitening agents of use include those described in US2008/34511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In another aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (CI Pigment Blue 29), UltramarineViolet (CA. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/l.

Encapsulates—The composition may comprise an encapsulate. In one aspect,an encapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. 85% or 90% of said encapsulates may have a fracturestrength of from 0.2 to 10 MPa, from 0.4 to 5MPa, from 0.6 to 3.5 MPa,or from 0.7 to 3MPa; and a benefit agent leakage of from 0 to 30%, from0 to 20%, or from 0 to 5%.

In one aspect, 85% or 90% of said encapsulates may have a particle sizefrom 1 to 80 microns, from 5 to 60 microns, from 10 to 50 microns, orfrom 15 to 40 microns.

In one aspect, 85% or 90% of said encapsulates may have a particle wallthickness from 30 to 250 nm, from 80 to 180 nm, or from 100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—In one aspect the composition comprises a perfume thatcomprises one or more perfume raw materials selected from the groupconsisting of 1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid,diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol,monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyI)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid,2-hydroxy-3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal,4-ethyl-.alpha.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-, [3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-;Undecanal, 2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone; 2-hydroxy-benzoic acid, methyl ester;2-hydroxy-benzoic acid, hexyl ester; 2-phenoxy-ethanol;2-hydroxy-benzoic acid, pentyl ester; 2,3-heptanedione; 2-hexen-1-ol;6-Octen-2-ol, 2,6-dimethyl-; damascone (alpha, beta, gamma or delta ormixtures thereof), 4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-,acetate; 9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificembodiments of the amphiphilic alkoxylated grease cleaning polymers ofthe present invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO91/08281 and PCT90/01815. Chemically, thesematerials comprise polyacrylates having one ethoxy side-chain per every7-8 acrylate units. The side-chains are of the formula —(CH₂CH₂O)_(m)(CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. The side-chains areester-linked to the polyacrylate “backbone” to provide a “comb” polymertype structure. The molecular weight can vary, but is typically in therange of 2000 to 50,000. Such alkoxylated polycarboxylates can comprisefrom 0.05 wt % to 10 wt % of the compositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate polymer—The composition of the present invention may alsoinclude one or more carboxylate polymers such as a maleate/acrylaterandom copolymer or polyacrylate homopolymer. In one aspect, thecarboxylate polymer is a polyacrylate homopolymer having a molecularweight of from 4,000 to 9,000Da, or from 6,000 to 9,000Da.

Soil release polymer—The composition of the present invention may alsoinclude one or more soil release polymers having a structure as definedby one of the following structures (I), (II) or (III):

—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)   (I)

—[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO—]_(e)   (II)

—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)   (III)

wherein:

-   a, b and c are from 1 to 200;-   d, e and f are from 1 to 50;-   Ar is a 1,4-substituted phenylene;-   sAr is 1,3-substituted phenylene substituted in position 5 with    SO₃Me;-   Me is Li, K, Mg/2, Ca/2, AI/3, ammonium, mono-, di-, tri-, or    tetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or    C₂-C₁₀ hydroxyalkyl, or mixtures thereof;-   R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or    C₁-C₁₈ n- or iso-alkyl; and-   R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched    C₂-C₃₀ alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a    C₈-C₃₀ aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic polymer—The composition of the present invention may alsoinclude one or more cellulosic polymers including those selected fromalkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose,alkyl carboxyalkyl cellulose. In one aspect, the cellulosic polymers areselected from the group comprising carboxymethyl cellulose, methylcellulose, methyl hydroxyethyl cellulose, methyl carboxymethylcellulose, and mixures thereof. In one aspect, the carboxymethylcellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 anda molecular weight from 100,000 to 300,000Da.

Enzymes—The composition may comprise one or more enzymes which providecleaning performance and/or fabric care benefits. Examples of suitableenzymes include, but are not limited to, hemicellulases, peroxidases,proteases, cellulases, xylanases, lipases, phospholipases, esterases,cutinases, pectinases, mannanases, pectate lyases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, malanases, ß-glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllasesand amylases, or mixtures thereof. A typical combination is an enzymecocktail that may comprise e.g. a protease and lipase in conjunctionwith amylase. When present in a composition, the aforementionedadditional enzymes may be present at levels from 0.00001 to 2 wt %, from0.0001 to 1 wt % or from 0.001 to 0.5 wt % enzyme protein by weight ofthe composition.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP0495257, EP0531372, WO96/11262, WO96/29397, WO98/08940.Other examples are cellulase variants such as those described inWO94/07998, EP0531315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254,WO95/24471, WO98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

In one aspect preferred enzymes would include a protease. Suitableproteases include those of bacterial, fungal, plant, viral or animalorigin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130,160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235,236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred thesubtilase variants may comprise the mutations: S3T, V4I, S9R, A15T,K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,RS103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A,G160D, Y167A, R170S, A194P, G195E, V199M, V2051, L217D, N218D, M222S,A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Preferenz™, Purafect MAO, Purafect Ox®, Purafect OxP®, Puramax®,Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® andOptimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequenceshown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (HenkelAG) and KAP (Bacillus alkalophilus subtilisin) from Kao. Suitablelipases and cutinases include those of bacterial or fungal origin.Chemically modified or protein engineered mutant enzymes are included.Examples include lipase from Thermomyces, e.g. from T. lanuginosus(previously named Humicola lanuginosa) as described in EP258068 andEP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipasefrom strains of Pseudomonas (some of these now renamed to Burkholderia),e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia(EP331376), P. sp. strain SD705 (WO95/06720 & WO96/27002), P.wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases(WO10/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinasefrom Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase fromThermobifida fusca (WO11/084412, WO13/033318, WO2013/096653),Geobacillus stearothermophilus lipase (WO11/084417), lipase fromBacillus subtilis (WO11/084599), and lipase from Streptomyces griseus(WO11/150157) and S. pristinaespiralis (WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4),including a bacterial polypeptide endogenous to a member of the genusBacillus which has a sequence of at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or even 100%identity to the amino acid sequence SEQID NO:2 in U.S. Pat. No. 7,141,403 and mixtures thereof. Suitableendoglucanases are sold under the tradenames Celluclean® and Whitezyme®(Novozymes).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco/Dupont).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Protected enzymes may beprepared according to the method disclosed in EP238216.

Dye Transfer Inhibiting Agents—The compositions of the present inventionmay also include one or more dye transfer inhibiting agents. Suitablepolymeric dye transfer inhibiting agents include, but are not limitedto, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a composition, the dye transfer inhibiting agents may bepresent at levels from 0.0001 to 10 wt %, from 0.01 to 5wt % or from 0.1to 3wt %.

Brighteners—The compositions of the present invention can also containadditional components that may tint articles being cleaned, such asfluorescent brighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75wt %, at least 90 wt%, at least 99wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making 0.1 fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. Nos. 4,790,856 and 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt %, from 0.05 wt %, from 0.1 wt % or from 0.2 wt % to upper levels of0.5 wt % or 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts—The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—The compositions of the present invention can also containdispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in compositions can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions.Examples of conventional stabilizing agents are, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example,WO92/19709 and WO92/19708 In case of aqueous compositions comprisingprotease, a reversible protease inhibitor, such as a boron compoundincluding borate, 4-formyl phenylboronic acid, phenylboronic acid andderivatives thereof, or compounds such as calcium formate, sodiumformate and 1,2-propane diol can be added to further improve stability.

Solvents—Suitable solvents include water and other solvents such aslipophilic fluids. Examples of suitable lipophilic fluids includesiloxanes, other silicones, hydrocarbons, glycol ethers, glycerinederivatives such as glycerine ethers, perfluorinated amines,perfluorinated and hydrofluoroether solvents, low-volatilitynonfluorinated organic solvents, diol solvents, otherenvironmentally-friendly solvents and mixtures thereof.

Structurant/Thickeners—Structured liquids can either be internallystructured, whereby the structure is formed by primary ingredients (e.g.surfactant material) and/or externally structured by providing a threedimensional matrix structure using secondary ingredients (e.g. polymers,clay and/or silicate material). The composition may comprise astructurant, from 0.01 to 5 wt %, or from 0.1 to 2.0 wt %. Thestructurant is typically selected from the group consisting ofdiglycerides and triglycerides, ethylene glycol distearate,microcrystalline cellulose, cellulose-based materials, microfibercellulose, hydrophobically modified alkali-swellable emulsions such asPolygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, andmixtures thereof. A suitable structurant includes hydrogenated castoroil, and non-ethoxylated derivatives thereof. A suitable structurant isdisclosed in U.S. Pat. No. 6,855,680. Such structurants have athread-like structuring system having a range of aspect ratios. Othersuitable structurants and the processes for making them are described inWO10/034736.

Conditioning Agents—The composition of the present invention may includea high melting point fatty compound. The high melting point fattycompound useful herein has a melting point of 25° C. or higher, and isselected from the group consisting of fatty alcohols, fatty acids, fattyalcohol derivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1 to 40 wt %, from 1 to 30 wt %, from 1.5 to 16wt %,from 1.5 to 8wt % in view of providing improved conditioning benefitssuch as slippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3wt %, from 0.075 to 2.0 wt %, or from 0.1to 1.0 wt %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH3 to pH9, or between pH4 and pH8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. Nos. 3,962,418; 3,958,581;and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. Nos. 5,104,646;5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837; 6,607,717;6,482,969; 5,807,956; 5,981,681; 6,207,782; 7,465,439; 7,041,767;7,217,777; US2007/0286837A1; US2005/0048549A1; US2007/0041929A1;GB849433; DE10036533, which are all incorporated herein by reference;Chemistry and Technology of Silicones, New York: Academic Press (1968);General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

The compositions of the present invention may also comprise from 0.05 to3 wt % of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. Nos. 5,674,478 and 5,750,122 or in 4,529,586; 4,507,280;4,663,158; 4,197,865; 4,217,914; 4,381,919; and 4,422,853.

Hygiene and malodour—The compositions of the present invention may alsocomprise one or more of zinc ricinoleate, thymol, quaternary ammoniumsalts such as Bardac®, polyethylenimines (such as Lupasol® from BASF)and zinc complexes thereof, silver and silver compounds, especiallythose designed to slowly release Ag⁺ or nano-silver dispersions.

Probiotics—The compositions may comprise probiotics such as thosedescribed in WO09/043709.

Suds Boosters—If high sudsing is desired, suds boosters such as theC₁₀-C₁₆ alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporatedinto the compositions, typically at 1 to 10 wt % levels. The C₁₀-C₁₄monoethanol and diethanol amides illustrate a typical class of such sudsboosters. Use of such suds boosters with high sudsing adjunctsurfactants such as the amine oxides, betaines and sultaines noted aboveis also advantageous. If desired, water-soluble magnesium and/or calciumsalts such as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added atlevels of, typically, 0.1 to 2 wt %, to provide additional suds and toenhance grease removal performance.

Suds Suppressors—Compounds for reducing or suppressing the formation ofsuds can be incorporated into the compositions of the present invention.Suds suppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. Nos. 4,489,455and 4,489,574, and in front-loading-style washing machines. A widevariety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See e.g. KirkOthmer Encyclopedia of Chemical Technology, Third Edition, Volume 7,p.430-447 (John Wiley & Sons, Inc., 1979). Examples of suds supressorsinclude monocarboxylic fatty acid and soluble salts therein, highmolecular weight hydrocarbons such as paraffin, fatty acid esters (e.g.,fatty acid triglycerides), fatty acid esters of monovalent alcohols,aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated amino triazines,waxy hydrocarbons preferably having a melting point below about 100° C.,silicone suds suppressors, and secondary alcohols. Suds supressors aredescribed in U.S. Pat. Nos. 2,954,347; 4,265,779; 4,265,779; 3,455,839;3,933,672; 4,652,392; 4,978,471; 4,983,316; 5,288,431; 4,639,489;4,749,740; 4,798,679; 4,075,118; EP89307851.9; EP150872; and DOS2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt%. Preferably, from 0.5 to 3wt % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2wt %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3wt %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain embodiments the compositions have cleaning activityfrom pH4 to pH11.5. In other embodiments, the compositions are activefrom pH6 to pH11, from pH7 to pH11, from pH8 to pH11, from pH9 to pH11,or from pH10 to pH11,5.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainembodiments, the optimum temperature range for the compositions is from10° C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20°C. to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C.to 45° C., or from 40° C. to 50° C.

Form of the Composition

The compositions described herein are advantageously employed forexample, in laundry applications, hard surface cleaning, dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. The compositions of the invention are in particular solidor liquid cleaning and/or treatment compositions. In one aspect theinvention relates to a composition, wherein the form of the compositionis selected from the group consisting of a regular, compact orconcentrated liquid; a gel; a paste; a soap bar; a regular or acompacted powder; a granulated solid; a homogenous or a multilayertablet with two or more layers (same or different phases); a pouchhaving one or more compartments; a single or a multi-compartment unitdose form; or any combination thereof.

The form of the composition may separate the components physically fromeach other in compartments such as e.g. water dissolvable pouches or indifferent layers of tablets. Thereby negative storage interactionbetween components can be avoided. Different dissolution profiles ofeach of the compartments can also give rise to delayed dissolution ofselected components in the wash solution.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids (US2009/0011970 A1).

Water-Soluble Film—The compositions of the present invention may also beencapsulated within a water-soluble film. Preferred film materials arepreferably polymeric materials. The film material can e.g. be obtainedby casting, blow-moulding, extrusion or blown extrusion of the polymericmaterial, as known in the art. Preferred polymers, copolymers orderivatives thereof suitable for use as pouch material are selected frompolyvinyl alcohols, polyvinyl pyrrolidone, polyalkylene oxides,acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters,cellulose amides, polyvinyl acetates, polycarboxylic acids and salts,polyaminoacids or peptides, polyamides, polyacrylamide, copolymers ofmaleic/acrylic acids, polysaccharides including starch and gelatine,natural gums such as xanthum and carragum. More preferred polymers areselected from polyacrylates and water-soluble acrylate copolymers,methylcellulose, carboxymethylcellulose sodium, dextrin, ethylcellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and most preferably selected from polyvinyl alcohols,polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC),and combinations thereof. Preferably, the level of polymer in the pouchmaterial, e.g. a PVA polymer, is at least 60 wt %. The polymer can haveany weight average molecular weight, preferably from about 1.000 to1.000.000, from about 10.000 to 300.000, from about 20.000 to 150.000.Mixtures of polymers can also be used as the pouch material.

Naturally, different film material and/or films of different thicknessmay be employed in making the compartments of the present invention. Abenefit in selecting different films is that the resulting compartmentsmay exhibit different solubility or release characteristics.

Preferred film materials are PVA films known under the MonoSol tradereference M8630, M8900, H8779 and those described in U.S. Pat. Nos.6,166,117 and 6,787,512 and PVA films of corresponding solubility anddeformability characteristics.

The film material herein can also comprise one or more additiveingredients. For example, it can be beneficial to add plasticisers, e.g.glycerol, ethylene glycol, diethyleneglycol, propylene glycol, sorbitoland mixtures thereof. Other additives include functional detergentadditives to be delivered to the wash water, e.g. organic polymericdispersants, etc.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. Nos. 4,762,636; 6,291,412;US20050227891A1; EP1070115A2; U.S. Pat. Nos. 5,879,584; 5,691,297;5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; 5,486,303 all ofwhich are incorporated herein by reference. The compositions of theinvention or prepared according to the invention comprise cleaningand/or treatment composition including, but not limited to, compositionsfor treating fabrics, hard surfaces and any other surfaces in the areaof fabric and home care, including: air care including air freshenersand scent delivery systems, car care, dishwashing, fabric conditioning(including softening and/or freshening), laundry detergency, laundry andrinse additive and/or care, hard surface cleaning and/or treatmentincluding floor and toilet bowl cleaners, granular or powder-formall-purpose or “heavy-duty” washing agents, especially cleaningdetergents; liquid, gel or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid types; liquid fine-fabricdetergents; hand dishwashing agents or light duty dishwashing agents,especially those of the high-foaming type; machine dishwashing agents,including the various tablet, granular, liquid and rinse-aid types forhousehold and institutional use: car or carpet shampoos, bathroomcleaners including toilet bowl cleaners; as well as cleaning auxiliariessuch as bleach additives and “stain-stick” or pre-treat types,substrate-laden compositions such as dryer added sheets. Preferred arecompositions and methods for cleaning and/or treating textiles and/orhard surfaces, most preferably textiles. The compositions are preferablycompositions used in a pre-treatment step or main wash step of a washingprocess, most preferably for use in textile washing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Method of Use

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. In one aspect of the invention, themethod comprises the step of contacting the surface to be treated in apre-treatment step or main wash step of a washing process, mostpreferably for use in a textile washing step or alternatively for use indishwashing including both manual as well as automated/mechanicaldishwashing. In one embodiment of the invention the lipase variant andother components are added sequentially into the method for cleaningand/or treating the surface. Alternatively, the lipase variant and othercomponents are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101958 and/or by applying alternating pressure(pressure/vaccum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one embodiment of Applicants' cleaning composition, cleaningadditive or mixture thereof. The fabric may comprise most any fabriccapable of being laundered in normal consumer or institutional useconditions. The solution may have a pH from 8 to 10.5. The compositionsmay be employed at concentrations from 500 to 15.000ppm in solution. Thewater temperatures typically range from 5° C. to 90° C. The water tofabric ratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using the polypeptidewith at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6 for producinga composition. In one aspect the invention relates to use of thecomposition for cleaning an object.

In one aspect the invention relates to a method of producing thecomposition, comprising adding a polypeptide with at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptideof SEQ ID NO: 2 or SEQ ID NO: 6, and a surfactant. In one aspect theinvention relates to a method for cleaning a surface, comprisingcontacting a lipid stain present on the surface to be cleaned with thecleaning composition. In one aspect the invention relates to a methodfor hydrolyzing a lipid present in a soil and/or a stain on a surface,comprising contacting the soil and/or the stain with the cleaningcomposition.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the polypeptidemay be used as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a polypeptide into the plant host genomeor chloroplast genome and propagating the resulting modified plant orplant cell into a transgenic plant or plant cell.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Media and Solutions

Unless otherwise indicated chemicals used as buffers and substrates werecommercial products of at least reagent grade. The commerciallyavailable enzymes Lipolase™ and Lipex™ were obtained from Novozymes A/S.Lipolase™ comprises the wildtype triacylglycerol lipase from Thermomyceslanuginosus expressed in Aspergillus oryzae. Lipex™ comprises atriacylglycerol lipase derived from the wildtype Thermomyces lanuginosustriacylglycerol lipase having the mutations T231R and N233R andexpressed in Aspergillus oryzae.

Strains

Escherichia coli Top-10 strain purchased from TIANGEN (TIANGEN BiotechCo. Ltd., Beijing, China) was used to propagate the expression vector.

Aspergillus oryzae MT3568 strain was used for heterologous expression ofthe gene encoding a polypeptide having homology with polypeptides withlipase activity. A. oryzae MT3568 is an amdS (acetamidase) disruptedgene derivative of A. oryzae JaL355 (WO02/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene with the pyrG gene.

Media

YPM media was composed of 10 g yeast extract, 20 g Bacto-peptone, 20 gmaltose, and deionized water to 1000 mL.

LB plates were composed of 10 g of Bacto-tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1000mL.

LB medium was composed of 10 g of Bacto-tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionized water to 1000 mL.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 mL of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes. Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCI, TritonX-100 (50 uL/500 mL) were added.

COVE-2 plate/tube for isolation: 30 g/L sucrose, 20 ml/L COVE saltsolution, 10 mM acetamide, 30 g/L noble agar (Difco, Cat #214220).

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 mL of COVE trace metal solution, and deionized water to1000 mL.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1000 mL.

Example 1 Identification and Cloning of a Lipase Gene from Actinomucorelegans (SEQ ID NO: 1

Chromosomal DNA from Actinomucor elegans was isolated by QIAamp DNABlood Mini Kit (Qiagen, Hilden, Germany). 5 ug of chromosomal DNA weresent for sequencing at FASTERIS SA, Switzerland. The genome sequenceswere analyzed for open reading frames encoding lipolytic enzymes and theActinomucor elegans lipase gene D145PS was identified. From the codingDNA sequence (CDS) of D145PS a protein sequence was translated (SEQ IDNO: 2). Base on the protein sequence of SEQ ID NO: 2 a synthetic CDSencoding this Actinomucor elegans lipase was designed (SEQ ID NO: 1)according to Aspergillus oryzae codon usage. This CDS was synthesisedand put into a pUC57 vector. For sub-cloning into an expression vector,this synthetic gene was amplified with the primers shown in Table 2.Upper characters represent the 5′- and 3′- regions of the genes to beamplified, while lower cases were homologous to the vector sequences atinsertion sites of pCaHj505 vector.

TABLE 1 Primers to amplify the synthetic Actinomucor elegans lipase geneSequence (5′-3′) Primer name SEQ ID acacaactggggatcc acc slip52200-NO: 3 ATGGTCTCCTTCACATTGATCTCCCA 5O_C505_BamHI SEQ IDgtcaccctctagatctcgag syn-pUC57-FLR NO: 4 AGCTATGACCATGATTACGCCAAGCT

For PCR amplification, 20 pmol of primer pair (each of the forward andreverse) were used in a PCR reaction composed of 1 uL of SEQ ID NO: 1comprising plasmid DNA, 10 uL of 5× GC Buffer, 1.5 uL of DMSO, 2.5 mMeach of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of Phusion™High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a finalvolume of 50 uL. The amplification was performed using a Peltier ThermalCycler (M J Research Inc., South San Francisco, Calif., USA) programmedfor denaturing at 98° C. for 1 minutes; 10 cycles of denaturing at 98°C. for 15 seconds, annealing at 65° C. for 30 seconds, with 1° C.decrease per cycle and elongation at 72° C. for 90 seconds; and another26 cycles each at 98° C. for 15 seconds, 60° C. for 30 seconds and 72°C. for 90 seconds; final extension at 72° C. for 10 minutes. The heatblock then went to a 4° C. soak cycle.

The PCR products were isolated by 0.7% agarose gel electrophoresis usingTBE buffer where the product band of 1.1 kb was visualized under UVlight. The PCR product was then purified from solution by using a GFXPCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire,UK).

Plasmid pCaHj505 (WO2013029496) was digested with BamHI and XhoI fromNEB (New England Biolabs, Frankfurt am Main Germany), and the resultingfragments were separated by 0.7% agarose gel electrophoresis using TBEbuffer, and purified using an GFX PCR DNA and Gel Band Purification Kit(GE Healthcare, Buckinghamshire, UK). 60 ng of this purified PCR productwere cloned into 200 ng of the previously digested expression vectorpCaHj505 by ligation with an IN-FUSION™ CF Dry-down Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA).

A 2.5 uL volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. 4 colonies were selected from LBagar plates containing 100 μg of ampicillin per ml and confirmed bycolony PCR with vector primers. The Actinomucor elegans lipase syntheticsequence was verified by DNA sequencing with vector primers (bySinoGenoMax Company Limited, Beijing, China). The plasmid comprising SEQID NO: 1 designated as D14CBC#1 was selected for protoplasttransformation and heterologous expression of its encoded lipase in anAspergillus oryzae host cell MT3568. The recombinant E.coli/transformant cells were cultivated overnight in 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. Plasmid DNA was purifiedusing a Qiagen Spin Miniprep kit (Cat. 27106) (QIAGEN GmbH, Hilden,Germany).

Using the SignalP program v.3 (Nielsen et al., 1997, Protein Engineering10: 1-6), a signal peptide of 27 residues was predicted. Using PEPSTATSfrom EMBOSS package (Rice et al., 2000, Trends in Genetics 16: 276-277)a mature protein of 356 amino acids (SEQ ID NO: 2) was predicted with amolecular mass of 39 kDa and an isoelectric point of 8.6.

Example 2 Transformation of Aspergillus oryzae with the Gene Encoding aLipase from Actinomucor elegans (SEQ ID NO: 1)

Protoplasts of Aspergillus oryzae MT3568 were prepared according toWO95/002043. 100 uL of protoplasts were mixed with 2.5-10 μg of theAspergillus expression vector comprising D14CBC#1 (Example 1) and 250 uLof 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 and gentlymixed. The mixture was incubated at 37° C. for 30 minutes and theprotoplasts were spread onto COVE sucrose plates for selection. Afterincubation for 4-7 days at 37° C. spores of 4 transformants wereinoculated into 3 ml of YPM medium. After 3 days cultivation at 30° C.,the culture broths were analyzed by SDS-PAGE using Novex® 4-20%Tris-Glycine Gel (Invitrogen Corporation, Carlsbad, Calif., USA) toidentify the transformants producing the largest amount of recombinantlipase from Actinomucor elegans.

The lipolytic activity produced by the Aspergillus transformants wasinvestigated using olive oil/agarose plates (1% protein grade agarose;1% olive oil; 0.008% brilliant green; 50 mM Hepes; pH 7.2). 20 uLaliquots of the culture broth from the different transformants, buffer(negative control), were distributed into punched holes with a diameterof 3 mm and incubated for 1 hour at 37° C. The plates were subsequentlyexamined for the presence or absence of a dark green zone around theholes corresponding to lipolytic activity.

Based on those two selection criteria, spores of the best transformantwere spread on COVE-2 plates for re-isolation in order to isolate singlecolonies. Then a single colony was spread on a COVE-2 tube untilsporulation.

Example 3 Fermentation of Aspergillus oryzae Transformed with the GeneEncoding a Lipase from Actinomucor elegans (SEQ ID NO: 1)

Spores from the best transformant were cultivated in 2400 mL of YPMmedium in shake flasks during 3 days at a temperature of 30° C. under 80rpm agitation. Culture broth was harvested by filtration using a 0.2 umfilter device. The filtered fermentation broth was precipitated by(NH₄)₂SO₄, and dialyzed with 20 mM Tris-HCl at pH7.0. Then the samplewas applied to Q Sepharose Fast Flow (GE Healthcare) equilibrated with20 mM Tris-HCl at pH7.0. A gradient of NaCl concentration was appliedfrom zero to 0.35M NaCl with 16 times column volume, then to 1M NaClwith 5 times column volume. The fractions with lipase activity werecollected and concentrated.

Example 4 Characterization of the Lipase (SEQ ID NO: 2)

The lipase as purified in the example above was characterized accordingto the following methods.

SDS-PAGE: Using SDS-PAGE the molecular weight the polypeptide havinglipase activity was shown to be about 40 kDa.

pNP-C8 assay: 4-Nitrophenyl octanoate from Sigma (C8:21742) wasdissolved in isopropanol in final concentration of 16.5 mM as stocksolution, then diluted 10 times with buffer at required pH, containing0.4% Triton X-100, 10 mM CaCl₂.

The reaction was started by mixing 20 μl enzyme sample at 0.5 mg enzymeprotein per ml or water as blank and 150 μl of the appropriate substrateworking solution, and incubation for the required time and read at 405nm.

pH Profile: 20 μl enzyme samples and 150 μl pNP-C8 in B&R buffer(Britton-Robinson buffer: 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mMCaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 3.0, 4.0, 5.0, 6.07.0, 8.0, 9.0 and 10.0 with HCl and NaOH) were mixed in an microtiterplate and placed on ice. The assay was initiated by transferring themicrotiter plate to an Eppendorf thermomixer set to 15° C. andincubating for 40 minutes, where after OD405 was read. The optimal pHfor this lipase is at around pH 8.0-9.0.

TABLE 2 pH Profile pH 3 4 5 6 7 8 9 10 Relative OD 5.34 2.21 7.21 0.880.34 100 44.6 −57.8

Temperature Profile: 20 uL enzyme samples and 150 uL pNP-C8 in Tris-HClat pH 7.0 were mixed in a microtiter plate and placed on ice. The assaywas initiated by transferring the microtiter plate to an Eppendorfthermomixer, which was set to an assay temperature of 15, 20, 30, 40,50, 60, 70 and 75° C. and incubating for 40 minutes where after OD405was read. The lipase has an optimal temperature of at least 75° C., anda broad temperature range with good activity from 30 up to at least 75°C.

TABLE 3 Temperature Profile Temperature (° C.) 15 20 30 40 50 60 70 75Relative OD 23 31 50 66 87 69 86.1 100

Thermostability: Samples of the enzyme was incubated at 50° C. for 0,10, 30, 60, and 120 minutes and placed on ice. 20 uL enzyme was addedinto 150 uL pNP-C8 solution with Tris-HCl at pH 8.0, incubated at 150°C. for 40 minutes and OD405 was read. The reactions were performed induplicate. The lipase is very stable at 50° C.

TABLE 4 Thermostability Time (mins) 0 10 30 60 120 Relative OD 100 110115 150 110

Determination of Td by Differential Scanning Calorimetry: Thethermostability of Acelip5(U1EC5) was determined by DifferentialScanning Calorimetry (DSC) using a VP-Capillary Differential ScanningCalorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermaldenaturation temperature, Td (° C.), was taken as the top ofdenaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer(50 mM Hepes, pH 8.0) at a constant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approx. 0.2 ml) were loaded into thecalorimeter (reference: buffer without enzyme) from storage conditionsat 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. priorto DSC scan from 20° C. to 100° C. Denaturation temperatures weredetermined at an accuracy of approximately +/−1° C. Td of the lipase ofthe invention at pH 8.0 was 59° C.

Example 5 Relative Wash Performance (SEQ ID NO: 2)

Washing experiments were performed using Automatic Mechanical StressAssay (AMSA) in order to assess the wash performance in laundry. TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid were vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted in glycine buffers at differentpH and in Model Detergents with different surfactant level. Theexperimental conditions are specified below:

Detergents/buffers: 50mM glycine buffer pH8

-   -   50 mM glycine buffer pH9    -   50 mM glycine buffer pH10    -   3.3 g/L Detergent 0% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 10% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 20% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 60% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 100% surfactant, 50mM glycine buffer pH8

-   Test solution volume: 160 uL

-   Wash time: 15 minutes

-   Temperature: 25° C.

-   Water hardness: 15° dH

-   Lipase dosage: 0 ppm or 1ppm

-   Test material: Cream turmeric stain according to WO06/125437

TABLE 5 Detergent composition (wt %) Total surfactant comprised 0% 10%20% 60% 100% NaOH, pellets (>99%) 0 0.18 0.35 1.05 1.75 Linearalkylbenzenesulfonic acid (LAS) (97%) 0 1.20 2.40 7.20 12.00 Sodiumlaureth sulfate (SLES) (28%) 0 1.76 3.53 10.58 17.63 Soy fatty acid(>90%) 2.75 2.75 2.75 2.75 2.75 Coco fatty acid (>99%) 2.75 2.75 2.752.75 2.75 AEO;alcohol ethoxylate with 8 mol EO;Lutensol TO 8 0 1.10 2.206.60 11.00 (~100%) Triethanol amine (100%) 3.33 3.33 3.33 3.33 3.33Na-citrate, dihydrate (100%) 2.00 2.00 2.00 2.00 2.00 DTMPA;diethylenetriaminepentakis(methylene) 0.48 0.48 0.48 0.48 0.48pentakis(phosphonic acid), heptasodium salt (Dequest 2066 C) (~42% asNa7 salt) MPG (>98%) 6.00 6.00 6.00 6.00 6.00 EtOH, propan-2-ol (90/10%)3.00 3.00 3.00 3.00 3.00 Glycerol (>99.5) 1.71 1.71 1.71 1.71 1.71Sodium formate (>95%) 1.00 1.00 1.00 1.00 1.00 PCA (40% as sodium salt)0.46 0.46 0.46 0.46 0.46 Water up to 100 100 100 100 100

Final adjustments to the specified pH were done with NaOH or citricacid. Water hardness was adjusted to 15° dH by addition of CaCl2 andMgCl₂ (Ca²+:Mg²⁺=4:1) to the test system.

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 85° C. for 5 min.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with turmeric. Turmericcontains the colorant curcumin, which function as a pH indicator byhaving pH dependent color change. Lipase activity leads to release offree fatty acids from the cream acylglycerides and this leads to pHdecrease and thereby color change of the curcumin pH indicator. Lipasewash performance can therefore be expressed as the extent of colorchange of light reflected-emitted from the washed soiled textile whenilluminated with white light.

Color measurements were made with a professional flatbed scanner (EPSONEXPRESSION 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark),which was used to capture an image of the washed soiled textile. Toextract a value for the light intensity from the scanned images, 24-bitpixel values from the image were converted into values for red, greenand blue (RGB).

Color change due to lipase activity was measured as the increase in thereflection-emitting of green light (G) relative to the sum ofreflected-emitted blue (B) and red (R) light. The wash performance(RP(Wash)) of a lipase relative to a reference lipase (LipexTM) wascalculated as: RP(Wash)=(G/(B+R)(tested lipase)−G/(B+R)(noenzyme))/(G/(B+R)(lipase ref.)−G/(B+R)(no enzyme)).

TABLE 6 Relative wash performance of the lipase of the invention (SEQ IDNO: 2) at various pH. Wash performance of Lipex ™ is 1.00. pH Lipase ofthe invention Buffer 0% surfactant 8 1.18 9 1.18 10 1.43 Detergent 0%surfactant 8 1.35 Detergent 10% surfactant 8 1.08 Detergent 20%surfactant 8 1.19 Detergent 60% surfactant 8 1.15 Detergent 100%surfactant 8 1.03

Example 6 Identification and Cloning of a Lipase Gene from Actinomucorelegans (SEQ ID NO: 5)

Chromosomal DNA from Actinomucor elegans was isolated by QIAamp DNABlood Mini Kit (Qiagen, Hilden, Germany). 5 pg of chromosomal DNA wereused for sequencing. The genome sequences were analyzed for open readingframes encoding lipolytic enzymes and the gene D145PQ of the Actinomucorelegans lipase of the invention was identified. From the coding DNAsequence (CDS) of D145PQ a protein sequence was translated (SEQ ID NO:6). Base on the protein sequence of SEQ ID NO: 6 a synthetic CDSencoding this Actinomucor elegans lipase was designed (SEQ ID NO: 5)according to Aspergillus oryzae codon usage. This CDS was put into apUC57 vector. For sub-cloning into an expression vector, this syntheticgene was amplified with the primers shown in Table 2. Upper charactersrepresent the 5′- and 3′-regions of the genes to be amplified, whilelower cases were homologous to the vector sequences at insertion sitesof pCaHj505 vector (WO2013029496).

TABLE 7 Primers to amplify the synthetic Actinomucor elegans lipase genePrimer name Sequence (5′-3′) SEQ ID acacaactggggatcc acc slip52200-NO: 7 ATGGTCTCCTTCGCCTCGATCC 2O_C505_BamHI SEQ ID gtcaccctctagatctcgagsyn-pUC57-FLR NO: 8 AGCTATGACCATGATTACGCCAAGCT

For PCR amplification, 20 pmol of primer pair (each of the forward andreverse) were used in a PCR reaction composed of 1 uL of SEQ ID NO: 5comprising plasmid DNA, 10 μl of 5× GC Buffer, 1.5 uL of DMSO, 2.5 mMeach of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of Phusion™High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a finalvolume of 50 uL. The amplification was performed using a Peltier ThermalCycler (M J Research Inc., South San Francisco, Calif., USA) programmedfor denaturing at 98° C. for 1 minutes; 10 cycles of denaturing at 98°C. for 15 seconds, annealing at 65° C. for 30 seconds, with 1° C.decrease per cycle and elongation at 72° C. for 90 seconds; and another26 cycles each at 98° C. for 15 seconds, 60° C. for 30 seconds and 72°C. for 90 seconds; final extension at 72° C. for 10 minutes. The heatblock then went to a 4° C. soak cycle.

The PCR products were isolated by 0.7% agarose gel electrophoresis usingTBE buffer where the product band of 1.1 kb was visualized under UVlight. The PCR product was then purified from solution by using a GFXPCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire,UK).

Plasmid pCaHj505 was digested with BamHI and XhoI from NEB (New EnglandBiolabs, Frankfurt am Main Germany), and the resulting fragments wereseparated by 0.7% agarose gel electrophoresis using TBE buffer, andpurified using an GFX PCR DNA and Gel Band Purification Kit (GEHealthcare, Buckinghamshire, UK). 60 ng of this purified PCR productwere cloned into 200 ng of the previously digested expression vectorpCaHj505 by ligation with an IN-FUSION™ CF Dry-down Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) according tothe manufacturer's instructions.

A 2.5 uL volume of the diluted ligation mixture was used to transform E.coli TOP-10 chemically competent cells. 4 colonies were selected from LBagar plates containing 100 μg of ampicillin per ml and confirmed bycolony PCR with vector primers. The Actinomucor elegans lipase syntheticsequence was verified by DNA sequencing with vector primers (bySinoGenoMax Company Limited, Beijing, China). The plasmid comprising SEQID NO: 5 designated as D14CB8#1 was selected for protoplasttransformation and heterologous expression of its encoded lipase in anAspergillus oryzae host cell MT3568. The recombinant E.coli/transformantcells were cultivated overnight in 3 mL of LB mediumsupplemented with 100 ug of ampicillin per mL. Plasmid DNA was purifiedusing a Qiagen Spin Miniprep kit (Cat. 27106) (QIAGEN GmbH, Hilden,Germany).

Using the SignalP program v.3 (Nielsen et al., 1997, Protein Engineering10: 1-6), a signal peptide of 41 residues was predicted. Using PEPSTATSfrom EMBOSS package (Rice et al., 2000, Trends in Genetics 16: 276-277)a mature protein of 337 amino acids (SEQ ID NO: 6) was predicted with amolecular mass of 38 kDa and an isoelectric point of 7.2.

Example 7 Transformation of Aspergillus oryzae with the Gene Encoding aLipase from Actinomucor elegans (SEQ ID NO: 5)

Protoplasts of Aspergillus oryzae MT3568 were prepared according toWO95/002043. 100 uL of protoplasts were mixed with 2.5-10 ug of theAspergillus expression l vector comprising D14CB8#1 (Example 6) and 250uL of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 and gentlymixed. The mixture was incubated at 37° C. for 30 minutes and theprotoplasts were spread onto COVE sucrose plates for selection. Afterincubation for 4-7 days at 37° C. spores of 4 transformants wereinoculated into 3 mL of YPM medium. After 3 days cultivation at 30° C.,the culture broths were analyzed by SDS-PAGE using Novex® 4-20%Tris-Glycine Gel (Invitrogen Corporation, Carlsbad, Calif., USA) toidentify the transformants producing the largest amount of recombinantlipase from Actinomucorelegans.

The lipoolytic activity produced by the Aspergillus transformants wasinvestigated using olive oil/agarose plates (1% protein grade agarose;1% olive oil; 0.008% brilliant green; 50 mM Hepes; pH7.2). 20 uLaliquots of the culture broth from the different transformants, buffer(negative control), were distributed into punched holes with a diameterof 3 mm and incubated for 1 hour at 37° C. The plates were subsequentlyexamined for the presence or absence of a dark green zone around theholes corresponding to lipolytic activity.

Based on those two selection criteria, spores of the best transformantwere spread on COVE-2 plates for re-isolation in order to isolate singlecolonies. Then a single colony was spread on a COVE-2 tube untilsporulation.

Example 8 Fermentation of Aspergillus oryzae Transformed with the GeneEncoding a Lipase from Actinomucor elegans (SEQ ID NO: 5)

Spores from the best transformant were cultivated in 2400 mL of YPMmedium in shake flasks during 3 days at a temperature of 30° C. under 80rpm agitation. Culture broth was harvested by filtration using a 0.2 umfilter device. The culture broth was precipitated by (NH₄)₂SO₄, anddialyzed with 20 mM Bis-Tris at pH6.0. Then the sample was applied to QSepharose Fast Flow (GE Healthcare) equilibrated with Bis-Tris at pH6.0.A gradient of NaCI concentration was applied from zero to 0.35M NaClwith 16 times column volume, then to 1M NaCl with 5 times column volume.The fractions with lipase activity were collected and concentrated.

Example 9 Characterization of the Lipase of the Invention (SEQ ID NO: 6)

The lipase as purified in the example was characterized according to thefollowing methods.

SDS-PAGE: Using SDS-PAGE the molecular weight the polypeptide havinglipase activity was shown to be about 30 kDa.

pNP-C8 assay: 4-Nitrophenyl octanoate from Sigma (C8:21742) wasdissolved in isopropanol in final concentration of 16.5 mM as stocksolution, then diluted 10 times with buffer at required pH containing0.4% Triton X-100, 10 mM CaCl₂. The reaction was started by mixing 20 uLenzyme sample at 0.5 mg/ml or water as blank and 150 uL of theappropriate substrate working solution, and incubation for the requiredtime and read at 405 nm.

pH Profile: 20 uL enzyme samples and 150 uL pNP-C8 in B&R buffer(Britton-Robinson buffer: 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mMCaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 3.0, 4.0, 5.0, 6.07.0, 8.0, 9.0 and 10.0 with HCl and NaOH) were mixed in an microtiterplate and placed on ice. The assay was initiated by transferring themicrotiter plate to an Eppendorf thermomixer set at 15° C. andincubating for 40 minutes, where after OD405 was read. The optimal pHfor the lipase of the invention is at around pH 8.0.

TABLE 8 pH Profile pH 3 4 5 6 7 8 9 10 Relative OD 5.9 3.19 6.35 4.063.86 100 49.3 −49.6

Temperature Profile: 20 uL enzyme samples and 150 uL pNP-C8 in Tris-HClat pH7.0 were mixed in a microtiter plate and placed on ice. The assaywas initiated by transferring the microtiter plate to an Eppendorfthermomixerset to the assay temperature at 15, 20, 30, 40, 50, 60, 70and 75 ° C. and incubating for 40 minutes. OD405 was read. The lipase ofthe invention works well at high temperatures, having an optimaltemperature of at least 75° C., but also shows good activity in therange from 30-75° C.

TABLE 9 Temperature Profile Temperature (° C.) 15 20 30 40 50 60 70 75Relative OD 28 30 49 67 81 77 84 100

Thermostability: Samples of the enzyme was incubated at 50° C. for 0,10, 30, 60, and 120 minutes and placed on ice. The 20 μl of the enzymesample was added into 150 uL pNP-C8 solution with Tris-HCl at pH8.0,incubated at 150° C. for 40 minutes and OD 405 was read. The lipase ofthe invention is surprisingly stable at 50° C. There was no activitydecrease after incubation for the longest tested period of 120 minutes.

TABLE 10 Thermostability Time (mins) 0 10 30 60 120 Relative OD 100 11092 97 99

Determination of Td by Differential Scanning Calorimetry: Thethermostability of the lipase of the invention was determined byDifferential Scanning Calorimetry (DSC) using a VP-CapillaryDifferential Scanning Calorimeter (MicroCal Inc., Piscataway, N.J.,USA). The thermal denaturation temperature, Td (° C.), was taken as thetop of denaturation peak (major endothermic peak) in thermograms (Cp vs.T) obtained after heating enzyme solutions (approx. 0.5 mg/mL) in buffer(50 mM Hepes, pH8.0) at a constant programmed heating rate of 200 K/hr.Sample- and reference-solutions (approx. 0.2 mL) were loaded into thecalorimeter (reference: buffer without enzyme) from storage conditionsat 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. priorto DSC scan from 20° C. to 100° C. Denaturation temperatures weredetermined at an accuracy of approximately +/−1° C. Td of the lipase ofthe invention at pH8.0 was 62° C.

Example 10 Relative Wash Performance (SEQ ID NO: 6)

Washing experiments were performed using Automatic Mechanical StressAssay (AMSA) in order to assess the wash performance in laundry. TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid were vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted in glycine buffers at differentpH and in Model Detergents with different surfactant level. Theexperimental conditions are specified below:

Detergents/buffers: 50mM glycine buffer pH8

-   -   50 mM glycine buffer pH9    -   50 mM glycine buffer pH10    -   3.3 g/L Detergent 0% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 0% surfactant, 50 mM glycine buffer pH9    -   3.3 g/L Detergent 10% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 10% surfactant, 50 mM glycine buffer pH9    -   3.3 g/L Detergent 20% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 20% surfactant, 50 mM glycine buffer pH9    -   3.3 g/L Detergent 60% surfactant, 50 mM glycine buffer pH8    -   3.3 g/L Detergent 60% surfactant, 50 mM glycine buffer pH9    -   3.3 g/L Detergent 100% surfactant, 50 mM glycine buffer pH8

-   Test solution volume: 160 uL

-   Wash time: 15 minutes

-   Temperature: 25° C.

-   Water hardness: 15° dH

-   Lipase dosage: Oppm or 0.5 ppm

-   Test material: Cream turmeric stain according to WO06/125437

TABLE 11 Detergent composition (wt %) Total surfactant comprised 0% 10%20% 60% 100% NaOH, pellets (>99%) 0 0.18 0.35 1.05 1.75 Linearalkylbenzenesulfonic acid (LAS) (97%) 0 1.20 2.40 7.20 12.00 Sodiumlaureth sulfate (SLES) (28%) 0 1.76 3.53 10.58 17.63 Soy fatty acid(>90%) 2.75 2.75 2.75 2.75 2.75 Coco fatty acid (>99%) 2.75 2.75 2.752.75 2.75 AEO;alcohol ethoxylate with 8 mol EO;Lutensol TO 8 0 1.10 2.206.60 11.00 (~100%) Triethanol amine (100%) 3.33 3.33 3.33 3.33 3.33Na-citrate, dihydrate (100%) 2.00 2.00 2.00 2.00 2.00 DTMPA;diethylenetriaminepentakis(methylene) 0.48 0.48 0.48 0.48 0.48pentakis(phosphonic acid), heptasodium salt (Dequest 2066 C) (~42% asNa7 salt) MPG (>98%) 6.00 6.00 6.00 6.00 6.00 EtOH, propan-2-ol (90/10%)3.00 3.00 3.00 3.00 3.00 Glycerol (>99.5) 1.71 1.71 1.71 1.71 1.71Sodium formate (>95%) 1.00 1.00 1.00 1.00 1.00 PCA (40% as sodium salt)0.46 0.46 0.46 0.46 0.46 Water up to 100 100 100 100 100

Final adjustments to the specified pH were done with NaOH or citricacid. Water hardness was adjusted to 15° dH by addition of CaCl₂ andMgCl₂ (Ca²+:Mg²⁺=4:1) to the test system.

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 85° C. for 5 min.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with turmeric. Turmericcontains the colorant curcumin, which function as a pH indicator byhaving pH dependent color change. Lipase activity leads to release offree fatty acids from the cream acylglycerides and this leads to pHdecrease and thereby color change of the curcumin pH indicator. Lipasewash performance can therefore be expressed as the extent of colorchange of light reflected-emitted from the washed soiled textile whenilluminated with white light.

Color measurements were made with a professional flatbed scanner (EPSONEXPRESSION 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark),which was used to capture an image of the washed soiled textile. Toextract a value for the light intensity from the scanned images, 24-bitpixel values from the image were converted into values for red, greenand blue (RGB).

Color change due to lipase activity was measured as the increase in thereflection-emitting of green light (G) relative to the sum ofreflected-emitted blue (B) and red (R) light. The wash performance(RP(Wash)) of a lipase relative to a reference lipase (LipexTM) wascalculated as: RP(Wash)=(G/(B+R)(tested lipase)−G/(B+R)(noenzyme))/(G/(B+R)(lipase ref.)−G/(B+R)(no enzyme)).

TABLE 12 Relative wash performance of the lipase of the invention (SEQID NO: 6) at various pH. Wash performance of Lipex ™ is 1.00. Lipase ofthe pH Lipolase ™ invention Buffer 0% surfactant 8 1.08 1.11 9 0.31 0.9810 0.23 0.50 Detergent 0% surfactant 8 1.04 1.21 9 0.44 1.03 Detergent10% surfactant 8 0.94 1.42 9 0.29 1.53 Detergent 20% surfactant 8 0.731.51 9 0.40 1.64 Detergent 60% surfactant 8 0.45 1.61 9 0.18 0.70Detergent 100% surfactant 8 0.31 1.50

1. A method, comprising contacting a lipid with a composition includingan isolated polypeptide that includes an amino acid sequence having atleast 85% sequence identity to the mature polypeptide of SEQ ID NO: 6,where the amino acid sequence has lipase activity.
 2. The method ofclaim 1, where the lipid is present on a textile or on a hard surface.3. The method of claim 1, where the isolated polypeptide includes anamino acid sequence having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 6, and where the amino acid sequence haslipase activity.
 4. The method of claim 1, where the compositionincludes at least one surfactant, at least one surfactant system, atleast one soap, or any mixtures thereof.
 5. The method of claim 4, wherethe surfactant or surfactant system is selected from anionicsurfactants, cationic surfactants, non-ionic surfactants, ampholyticsurfactants, zwitterionic surfactants, semipolar nonionic surfactantsand any mixtures thereof.
 6. The method of claim 5, where the surfactantor surfactant system includes linear alkylbenzenesulfonates (LAS). 7.The method of claim 5, where the surfactant or surfactant systemincludes alcohol ethoxysulfates (AES).
 8. The method of claim 5, wherethe surfactant or surfactant system includes alcohol ethoxylates (AE).9. The method of claim 5, where the surfactant or surfactant systemincludes sodium dodecyl benzene sulfonate, sodium hydrogenated cocoate,sodium laureth sulfate, C12-14 pareth-7, C12-15 pareth-7, sodium C12-15pareth sulfate, or C14-15 pareth-4.
 10. The method of claim 5, where thesurfactant or surfactant system is present at a level of from 0.1 to 60wt %.
 11. The method of claim 1, where the composition is a laundrycleaning composition, a dishwashing cleaning composition, a hard-surfacecleaning composition or a personal care cleaning composition.
 12. Themethod of claim 1, where the isolated polypeptide includes the maturepolypeptide of SEQ ID NO: 6.